TY - JOUR
T1 - A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution
AU - Hidalgo, Aurelio
AU - Schliessmann, Anna
AU - Molina, Rafael
AU - Hermoso, Juan
AU - Bornscheuer, Uwe T
PY - 2008/9
Y1 - 2008/9
N2 - Protein engineering is currently performed either by rational design, focusing in most cases on only a few positions modified by site-directed mutagenesis, or by directed molecular evolution, in which the entire protein-encoding gene is subjected to random mutagenesis followed by screening or selection of desired phenotypes. A novel alternative is focused directed evolution, in which only fragments of a protein are randomised while the overall scaffold of a protein remains unchanged. For this purpose, we developed a PCR technique using long, spiked oligonucleotides, which allow randomising of one or several cassettes in any given position of a gene. This method allows over 95% incorporation of mutations independently of their position within the gene, yielding sufficient product to generate large libraries, and the possibility of simultaneously randomising more than one locus at a time, thus originating recombination. The high efficiency of this method was verified by creating focused mutant libraries of Pseudomonas fluorescens esterase I (PFEI), screening for altered substrate selectivity and validating against libraries created by error-prone PCR. This led to the identification of two mutants within the OSCARR library with a 10-fold higher catalytic efficiency towards p-nitrophenyl dodecanoate. These PFEI variants were also modelled in order to explain the observed effects.
AB - Protein engineering is currently performed either by rational design, focusing in most cases on only a few positions modified by site-directed mutagenesis, or by directed molecular evolution, in which the entire protein-encoding gene is subjected to random mutagenesis followed by screening or selection of desired phenotypes. A novel alternative is focused directed evolution, in which only fragments of a protein are randomised while the overall scaffold of a protein remains unchanged. For this purpose, we developed a PCR technique using long, spiked oligonucleotides, which allow randomising of one or several cassettes in any given position of a gene. This method allows over 95% incorporation of mutations independently of their position within the gene, yielding sufficient product to generate large libraries, and the possibility of simultaneously randomising more than one locus at a time, thus originating recombination. The high efficiency of this method was verified by creating focused mutant libraries of Pseudomonas fluorescens esterase I (PFEI), screening for altered substrate selectivity and validating against libraries created by error-prone PCR. This led to the identification of two mutants within the OSCARR library with a 10-fold higher catalytic efficiency towards p-nitrophenyl dodecanoate. These PFEI variants were also modelled in order to explain the observed effects.
KW - Amino Acid Sequence
KW - Bacterial Proteins/chemistry
KW - Carboxylesterase/chemistry
KW - Directed Molecular Evolution
KW - Gene Library
KW - Models, Molecular
KW - Molecular Sequence Data
KW - Mutagenesis, Site-Directed
KW - Oligonucleotides/metabolism
KW - Polymerase Chain Reaction
KW - Pseudomonas fluorescens/enzymology
KW - Substrate Specificity
U2 - 10.1093/protein/gzn034
DO - 10.1093/protein/gzn034
M3 - Journal article
C2 - 18559369
SN - 1741-0126
VL - 21
SP - 567
EP - 576
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 9
ER -